Anti-angiogenic peptides and uses thereof

ABSTRACT

Provided are peptide sequences derived from prostate serum antigen (PSA). The peptides are provided in cyclic and linear form. Methods for using the peptides for inhibition of angiogenesis, such as angiogenesis in a tumor, are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationNo. 61/513,019, filed Jul. 29, 2011, the disclosure of which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to cancer therapy and morespecifically to novel peptides for use as anti-angiogenic/anti-canceragents.

BACKGROUND OF THE INVENTION

Prostate cancer is a major contributor to cancer mortality in Americanmales causing projected death of approximately 27,360 men in 2009 (Jemalet al., Cancer J. Clin., 59: 225-249, 2009). Therapeutic modalities suchas radical prostatectomy and radiotherapy are sometimes curative forlocalized disease, yet no treatments for metastatic prostate cancer thatsignificantly increases patient survival are available.

Angiogenesis is critical in tumor progression and metastasis in most ifnot all solid tumors, since a functional vascular supply is required forthe continued growth of solid tumors and the spread of cancer cells.Small non-growing tumors may remain dormant for years and the angiogenicswitch to aggressive metastatic phenotype involves a change in the localequilibrium between factors inducing blood vessel formation and thoseinhibiting the process.

There is an ongoing and unmet need to develop compositions and methodsfor inhibiting angiogenesis as a therapeutic modality for treating solidtumors, and in particular, for prophylaxis and/or therapy of prostatetumors. The present invention meets this need.

SUMMARY OF THE INVENTION

The present invention provides peptides and methods of using thepeptides for inhibiting angiogenesis. Thus, the invention providescompositions and methods for use in therapeutic interventions fortreating diseases that include but are not limited to cancer.

The peptides of the invention include but are not limited to peptidescomprising or consisting of amino acid sequences disclosed herein,fragments of the amino acid sequences, and modifications of peptidescomprising amino acid sequences derived from prostate specific antigen(PSA). Each peptide and peptide that can be modified as described hereinand which is encompassed within the scope of the invention has thecapability to inhibit human endothelial cell migration, such asendothelial cell migration in a gelatinous protein mixture, such as thatsold under the name Matrigel. Thus, in one embodiment, a peptideencompassed within the scope of the invention is a peptide that caninhibit cell network formation in a gelatinous protein mixture.

In particular embodiments, the amino acids of the peptides provided bythe invention comprise or consist of the following sequences:

Lys Asn Arg Phe Leu Arg Pro Gly Asp Asp Ser Ser His (SEQ ID NO:1). Thispeptide is also referred to herein as Peptide #1 (linear) and as“PSA-P1L.” It is not provided as a cyclic peptide. A modified version ofthis peptide is provided and is referred to herein as PSA-P1C(constrained, or cyclized). It has the sequence Cys Lys Asn Arg Phe LeuArg Pro Gly Asp Asp Ser Ser His Cys (SEQ ID NO:7). In this peptide, acysteine was engineered at the N-terminus and at the C-terminus ofPeptide #1. These facilitate formation of a disulfide bond between theN- and C-terminal cysteine residues. The result is a cyclic(“constrained” or cyclized) peptide wherein the N- and C-termini areconnected to one another via a disulfide bond.

In another embodiment, Peptide #2 is provided. It has the sequence GlyTrp Gly Ser Ile Glu Pro Glu Glu Phe Leu Thr Pro Lys Lys Leu Gln (SEQ IDNO:2).

In another embodiment, Peptide #3 is provided. It has the sequence AsnAsp Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys (SEQ ID NO:3).

In another embodiment, Peptide #3 is modified to replace the Cys that isendogenous to PSA with Ala, to provide Peptide 3*. Peptide 3* has thesequence Asn Asp Val Ala Ala Gln Val His Pro Gln Lys Val Thr Lys (SEQ IDNO:8).

In another embodiment, Peptide #4 is provided. It has the sequence GlyArg Trp Thr Gly Gly Lys Ser Thr Cys Ser (SEQ ID NO:4).

In another embodiment, Peptide #5 is provided. It has the sequence SerGlu Pro Cys Ala Leu Pro Glu Arg Pro Ser (SEQ ID NO:5).

In another embodiment, Peptide #6 is provided. It has the sequence AsnAsp Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys Phe Met Leu Cys Ala(SEQ ID NO:6). Peptide #6 is also referred to herein as “PSA-P3C”. Thetwo Cys in PSA-P3C can cyclize (constrain) the peptide via a disulfidebond. In one embodiment, Peptide #6 is modified so that the two Cysresidues in it are replaced by Ala to provide a peptide which has thesequence Asn Asp Val Ala Ala Gln Val His Pro Gln Lys Val Thr Lys Phe MetLeu Ala Ala (SEQ ID NO:9). This peptide is referred to herein as“PSA-P3L” and is not provided in cyclic form.

The method comprises administering a composition comprising one or morepeptides comprising or consisting of an amino acid sequence as describedherein to an individual in an amount effective to inhibit angiogenesis,and/or in an amount effective to inhibit the growth and/or metastasis ofa tumor. Other diseases that involve undesirable or irregularangiogenesis are also contemplated for treatment. Compositionscomprising the peptides are also provided. Such compositions include butare not necessarily limited to pharmaceutical preparations.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 top, is a graphical depiction of PSA-induced alteration ofexpression of select genes in PC-3M cells. The affected genes in thepanels in FIG. 1 top are: Panel A (Pim-1), Panel B (VEGF), Panel C(EphA2), Panel D (uPA), Panel E (Bcl2), Panel F (IFN-γ). FIG. 1, bottom,is a representation of a functional-Magnetic Resonance Image (f-MRI)analysis of PSA-induced inhibition of growth of PC-3M xenografts.

FIG. 2 is a photographic representation and table showing PSA-mediatedprotein modulation in PC-3M cells as determined by 2-dimensionaldifference gel electrophoresis (2D-DIGE) analysis.

FIG. 3 is a graphical representation of data obtained from quantitativereal time polymerase chain reaction (Q-PCR) analysis of PSA-mediatedmodulation of gene expression in Human umbilical vein endothelial(HUVEC) treated with 10 μM PSA using Peptide No. 3.

FIG. 4 is a graphical representation of data demonstrating thatenzymatic activity of purified PSA (f-PSA) is not required for in vitroanti-angiogenic activity.

FIG. 5 is a graphical representation of data demonstrating effects ofenzymatically active and enzymatically inactive f-PSA on gene expressionin HUVEC cells.

FIG. 6 is a graphical representation of data demonstrating effects ofenzymatically active and enzymatically inactive f-PSA on expression ofgenes involved in angiogenesis in HUVEC.

FIG. 7 is a photographic representation of Matrigel Endothelial TubeFormation Assay. NT=non-tumor tissue and SP=seminal plasma. Both typesare intact and enzymatically active.

FIG. 8 is 3-Dimensional model of human PSA and depicts the location offive hydrophilic peptide sequences tested as described herein foranti-angiogenic activity.

FIG. 9 is a graphical representation of data showing anti-angiogenicactivity of five candidate PSA-mimetic peptides in the HUVEC tubeformation assay. (Peptide 3 isasn-asp-val-ala-ala-gln-val-his-pro-gln-lys-val-thr-lys (SEQ ID NO: 8)).

FIG. 10 is a graphical representation of data showing anti-angiogenicactivity of Peptides 1 (SEQ ID NO:1) and 3*(SEQ ID NO:8) relative to theanti-angiogenic activity of native PSA.

FIG. 11 is a graphical representation of data showing anti-angiogenicactivity of a linear peptide (PSA-P3L) of the invention versus a cyclicpeptide (PSA-P3C) of the invention.

FIG. 12 depicts mass spectroscopy analysis of selected proteins andsuggests that by identifying cell membrane proteins that bind to PSA byco-precipitation, it will be possible to identify the receptor for PSAthat transmits the signal into the cell that changes gene transcriptionand angiogenic activity. The data were obtained by treatment of HUVECwith biotinylated-PSA at 4C to identify the ligand(s) that may be thepart of receptor. Two targets were identified: 40S ribosomal protein SAand intracellular adhesion molecule 2 (ICAM-2).

FIG. 13 is a graphical representation of data showing anti-angiogenicactivity of f-PSA, PSA-P3L, and PSA-P3C by inhibiting tube formation byHUVEC in a Matrigel assay.

FIG. 14 is a graphical representation of data showing of f-PSA, PSA-ACTComplex, PSA-P3L, and PSA-P3L-ACT complex by inhibiting tube formationby HUVEC in a Matrigel assay.

FIG. 15 is a graphical representation of data showing modulation of FLT1gene expression in HUVEC by f-PSA and PSA-P31.

FIG. 16 is a graphical representation of data showing modulation of FAKgene expression in HUVEC by f-PSA and PSA-P3L.

FIG. 17 is a graphical representation of data showing modulation of VEGFgene expression in HUVEC by f-PSA, PSA-P3L and PSA-P3C.

FIG. 18 is a graphical representation of data showing modulation of ANG2gene expression in HUVEC by f-PSA, PSA-P31 and PSA-P3C.

FIG. 19 is a graphical representation of data showing anti-angiogenicactivity of f-PSA, PSA-P1L and PSA-P3L via inhibition of tube formationby HUVEC in Matrigel.

FIG. 20 is a graphical representation of data showing anti-angiogenicactivity of f-PSA and PSA-ACT complex via inhibition of tube formationby HUVEC in Matrigel.

FIG. 21 is photographic representation of the effect of f-PSA, PSA-P1L,PSA-P1C, PSA-P3L and PSA-P3C on migratory properties of HUVEC.

DESCRIPTION OF THE INVENTION

The present invention provides isolated peptides derived from PSA thatare expected to be capable of inhibiting angiogenesis in vivo,compositions comprising the peptides, and methods of using the peptidesto inhibit angiogenesis.

In more detail, PSA (also referred to in the art as KLK3) is widelyrecognized by the lay population as the serum biomarker utilized forscreening for the presence, aggressiveness and/or recurrence of prostatecancer. However, recent observations have called into question theutility of PSA as a screening or diagnostic tool, and the biology of PSAproduction and function in the human prostate is not consistent with acausal role for PSA in the progression of prostate cancer. PSA is at10⁶-fold greater levels in seminal fluid, and 10³-fold greater levels inthe prostate tissue, than the level of PSA present in the serum ofpost-pubertal males. The PSA in seminal fluid and prostate tissue islargely enzymatically active and is not complexed with chaperone/carrierproteins, whereas, PSA in serum is largely enzymatically inactive andsequestered through binding to chaperone/carrier proteins. Furthermore,prostate production of PSA falls with age, prostate cancer progressionand androgen deprivation therapy, whereas, the serum levels of PSAmeasured in the screening test rise even though prostate production ofPSA is falling.

PSA was cloned and characterized as a product of the human prostategland, however, PSA is produced by many organs in the human, in bothmales and females. In females, PSA is produced in benign breast,endometrial and ovarian tissues, and malignancies of these tissues, aswell as in non-hormonally regulated tissues and their cancers, such asthe parotid gland. Across all of these malignancies, including prostatecancer, tissue levels of PSA in the cancer tissue are inverselycorrelated with prognosis.

PSA is well known to undergo proteolytic processing from a full lengthprotein that initially contains a signal peptide sequence (residues 1-17of the full length protein), a sequence corresponding to an activationpeptide (residues 18-24), and the processed PSA protein, whichordinarily consists of 237 amino acids spanning residues 25-261 of thefull length protein. The 237 amino acid protein (25-261) were used inthe studies that include PSA as described herein. The full length (i.e.,unprocessed) protein consists of the amino acid sequence:

(SEQ ID NO: 10) MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP.

The principal biological function of PSA is currently believed to be inliquification of seminal fluid. PSA has also been shown to havesignificant transcriptional regulatory activity, modulating expressionof multiple potent anti-angiogenic genes and processes. The presentinvention provides evidence that the transcriptional regulatory,anti-tumorigenic, and anti-angiogenic activity of human PSA areindependent of the enzymatic activity of PSA, in that these biologicalendpoints are observed at equal molar concentrations of enzymaticallyinactive PSA. We demonstrate using established techniques for evaluatinganti-angiogenic agents that the anti-angiogenic activity is exhibited bysmall peptides that we have derived from human PSA. Thus, the presentinvention provides a plurality of peptides derived from PSA, each ofwhich is expected to be useful for inhibition of angiogenesis, eitheralone, or in combination with each other. Each peptide and derivative(s)thereof that are encompassed within the scope of the present inventionhas the capability to inhibit human endothelial cell migration in agelatinous protein mixture. Thus, the invention provides a method ofinhibiting human endothelial cell migration comprising contacting humanendothelial cells with a composition comprising a peptide describedherein, wherein the migration is inhibited subsequent to the contactingof the human endothelial cells with the composition. Also provided is amethod of inhibiting angiogenesis and/or tumor growth and/or metastasis.The method comprises administering a composition comprising one or moreof the peptides of the invention to an individual in an amount effectiveto inhibit angiogenesis, and/or inhibit tumor growth and/or inhibitmetastasis. Also provided pharmaceutical preparations comprising thepeptides, which can be used in the methods of the invention.

Without intending to be bound by any particular theory, it is consideredthat PSA is an endogenous anti-neoplastic factor in many human tissuesand their malignancies, and that the peptides described herein willprovide anti-angiogenic activity targeted to the immature/angiogenictumor vascular network, thus inhibiting angiogenesis, and/or tumorprogression and/or metastasis. The peptides described herein arebelieved to represent ideal therapeutic modalities in that they are aderived from a constitutively expressed protein that should not elicitan immune response, and systemic administration is expected to providedirect delivery to the therapeutic target, namely, the endothelial cellsof the cancer.

Data presented herein show that a subset of synthetic peptides derivedfrom human PSA inhibit angiogenesis in the Matrigel endothelial celltube formation assay. It is noteworthy that tube formation in Matrigelby cultured endothelial cells, usually HUVEC, is an established in vitrosurrogate for angiogenesis that is proposed to require both migrationand differentiation by the endothelial cells. The “tube formation” assayhas been utilized extensively to evaluate in vitro the anti-angiogeniccapacity of peptides, proteins and pharmacologic agents (Print, C., etal., Soluble factors from human endometrium promote angiogenesis andregulate the endothelial cell transcriptome. Hum Reprod, 2004. 19(10):p. 2356-66; Delves, G. H., et al., In vitro inhibition of angiogenesisby prostasomes. 2005. Prostate Cancer Prostatic Dis. 8(2): p. 174-8).Thus, results presented herein are strongly indicative that the peptidesof the invention will be suitable for inhibition of angiogenesis, andthus can be expected to be useful for prophylaxis and/or therapy ofhuman cancers, including but not limited to prostate cancer, breastcancer, ovarian cancer, cervical cancer and parotid cancers. In certainembodiments, the invention provides a method for inhibiting themigration and/or differentiation of human endothelial cells comprisingcontacting the cells with a composition comprising one or more peptidesof the invention.

TABLE 1 PSA levels in paired tumor and non-tumor tissue Tumor tissueNon-tumor tissue Donor [ng PSA/ug DNA] [ng PSA/ug DNA] 1. 1759 2118 2.918 2270 3. 1027 1796 4. 359 777 5. 303 1435 6. 504 1856 7. 815 2017 8.1023 2491 9. 1159 1408 10. 1333 1921 11. 224 879 12. 414 862 13. 4401660 14. 659 1440 15. 448 1095 Average 759 1601

The data in Table #1 shows that PSA levels in prostate gland asexpressed in prostate cancer tissue are lower than in benign tissue inthe same gland. At the time of surgery, prostate tissue, that isremoved, is checked by pathology to determine what portion of tissue isbenign and what portion is malignant. Thus, Table 1 establishes thattissue PSA is down regulated in cancerous tissue. The significance ofthe data presented in this Table, without intending to be bound by anyparticular theory, is that since it is generally accepted that serum PSAlevels increase in prostate cancer, it is not a logical hypothesis thatPSA is an endogenous antiangiogenic factor. However, we have determinedthat malignant tissues actually make less PSA than normal tissue (asdescribed in the Table) which actually reduces expression of theendogenous antiangiogenic agent.

The following peptide sequences illustrate embodiments of peptidesencompassed by the present disclosure. It is notable that Peptides #2,#4 and #5 did not demonstrate reproducible anti-angiogenic activity.Thus, not all fragments of PSA exhibit anti-angiogenic properties.

Lys Asn Arg Phe Leu Arg Pro Gly Asp Asp Ser Ser His (SEQ ID NO:1). Thispeptide is also referred to in the description of the invention and theExamples as Peptide #1 and as “PSA-P1L.” This peptide is not cyclized.We modified this peptide to provide the peptide referred to herein as“PSA-P1C.” It has the sequence Cys Lys Asn Arg Phe Leu Arg Pro Gly AspAsp Ser Ser His Cys (SEQ ID NO:7). To derive PSA-P1C, we engineered acysteine at the N-terminus and at the C-terminus of Peptide #1, whichenabled formation of a disulfide between the N- and C-terminal residues.The result is a cyclic peptide wherein the N- and C-termini areconnected to one another via a disulfide bond. In all cases wherePSA-P1L is tested in the data presented herein it was tested in thecyclic form.

Peptide #2 is provided and has the sequence Gly Trp Gly Ser Ile Glu ProGlu Glu Phe Leu Thr Pro Lys Lys Leu Gln (SEQ ID NO:2).

In another embodiment, Peptide #3 is provided and has the sequence AsnAsp Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys (SEQ ID NO:3).

We modified Peptide #3 to replace the Cys that is endogenous to PSA withAla, resulting in Peptide 3*. Peptide 3* has the sequence Asn Asp ValAla Ala Gln Val His Pro Gln Lys Val Thr Lys (SEQ ID NO:8).

Peptide #4 has the sequence Gly Arg Trp Thr Gly Gly Lys Ser Thr Cys Ser(SEQ ID NO:4). Peptide #5 has the sequence Ser Glu Pro Cys Ala Leu ProGlu Arg Pro Ser (SEQ ID NO:5).

In another embodiment, Peptide #6 is provided. It has the sequence AsnAsp Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys Phe Met Leu Cys Ala(SEQ ID NO:6). Peptide #6 is also referred to herein as “PSA-P3C.” Thetwo Cys in PSA-P3C are used to cyclize the peptide via a disulfide bond.PSA-P3C was tested in the experiments described herein in cyclic form.We modified Peptide #6 to test a similar peptide in linear form. To dothis, the two Cys residues in Peptide #6 were replaced by Ala to providea peptide which has the sequence Asn Asp Val Ala Ala Gln Val His Pro GlnLys Val Thr Lys Phe Met Leu Ala Ala (SEQ ID NO:9). This peptide isreferred to herein as “PSA-P3L”.

It is expected that the amino acids sequences of the peptides presentedherein and described as suitable for use in the invention can bemodified and still retain desirable properties, such as anti-angiogenicproperties. Such modifications can be determined in a variety of ways,such as by almandine scanning mutagenesis. Illustrative, non-limitingexamples of peptide derivatives based upon Peptide #1 that can beproduced in this manner, and are therefore included within the scope ofthe invention, are illustrated by the following sequences:

(SEQ ID NO: 11) lys ala arg phe leu arg pro gly asp asp   ser ser his(SEQ ID NO: 12) lys asn ala phe leu arg pro gly asp asp  ser ser his(SEQ ID NO: 13) lys asn arg ala leu arg pro gly asp asp  ser ser his(SEQ ID NO: 14) lys asn arg phe ala arg pro gly asp asp   ser ser his(SEQ ID NO: 15) lys asn arg phe leu ala pro gly asp asp   ser ser his(SEQ ID NO: 16) lys asn arg phe leu arg ala gly asp asp   ser ser his(SEQ ID NO: 17) lys asn arg phe leu arg pro ala asp asp   ser ser his(SEQ ID NO: 18) lys asn arg phe leu arg pro gly ala asp   ser ser his(SEQ ID NO: 19) lys asn arg phe leu arg pro gly asp ala   ser ser his(SEQ ID NO: 20) lys asn arg phe leu arg pro gly asp asp   ala ser his(SEQ ID NO: 21) lys asn arg phe leu arg pro gly asp asp  ser ala his(SEQ ID NO: 22) lys asn arg phe leu arg pro gly asp asp   ser ser ala

The invention includes peptides of various lengths, and with variousamino acid substitutions, based on the amino acid sequences of thepeptides provided by the invention. For example, Peptide #1 (or anyother peptide described herein) can be modified by conservative aminoacid substitutions that are based generally on relative similarity of R—group substituents. Non-limiting examples of such substitutionscontemplated in the present invention include, but are not limited to:gly or ser for ala; lys for arg; gln or his for asn; glu for asp; serfor cys; asn for gln; asp for glu; ala for gly; asn or gln for his; leuor val for ile; ile or val for leu; arg for lys; leu or tyr for met; thrfor ser; tyr for trp; phe for tyr; and ile or leu for val. Thus,peptides that comprise any single conservative amino acid substitution,or any combination of conservative amino acid substitutions, areincluded in the invention, so long as they retain their anti-angiogenicproperties. In certain embodiments, peptides of the invention whichcomprise conservative amino acid substitutions retain their capabilityto inhibit tubule formation by HUVECs.

Non-conservative substitutions that enhance desirable characteristics ofthe peptides, such as their anti-angiogenic activity, circulation time,bioavailability, stability, binding to cell surface receptors,regulation of gene transcription, targeting imaging or therapeuticmodalities to the tumor vasculature, etc., are also included in theinvention.

In addition to amino acid substitutions, peptides of the invention mayinclude fragments of any of the peptides described herein. Any peptideencompassed within the scope of the invention can comprise or consist offrom 4-20 amino acids, inclusive, and including all integers therebetween. For example, Peptide #1 fragments encompassed within the scopeof the invention include but are not limited to:

(SEQ ID NO: 23) asn-arg-phe-leu-arg-pro-gly-asp-asp-ser-ser-his(SEQ ID NO: 24) arg-phe-leu-arg-pro-gly-asp-asp-ser-ser-his (SEQ ID NO: 25) phe-leu-arg-pro-gly-asp-asp-ser-ser-his (SEQ ID NO: 26)leu-arg-pro-gly-asp-asp-ser-ser-his  (SEQ ID NO: 27)arg-pro-gly-asp-asp-ser-ser-his  (SEQ ID NO: 28)pro-gly-asp-asp-ser-ser-his (SEQ ID NO: 29) gly-asp-asp-ser-ser-his (SEQ ID NO: 30) asp-asp-ser-ser-his  (SEQ ID NO: 31) asp-ser-ser-his Each peptide described herein can also be truncated by any number ofamino acids at its C-terminus, and by any number of amino acids at boththe N- and C-termini, so long as the remaining sequence retains itsanti-angiogenic activity. Each of the foregoing fragments can also besubjected to the aforementioned amino acid substitutions, and thus,fragments having such substitutions are included within the scope of theinvention. Any peptide provided by the invention can comprise one, two,three, four, or five conservative amino acid substitutions. Any of thepeptides provided by the invention can be at least four amino acids inlength.

To the extent that any of the peptides/fragment sequences per se havebeen previously described, then the present invention contemplates theiruse in methods for inhibition of angiogenesis, and/or for theprophylaxis and/or treatment of tumors and/or metastasis, and inpharmaceutical prepartions. It is also contemplated that the peptides ofthe present invention may include additional amino acids, and mayinclude modified amino acids that can improve any desirable property ofthe peptides. Thus, the peptides could be covalently or non-covalentlyassociated with any desirable moiety that would be expected to improvetheir functional capabilities in accordance with the method of theinvention.

The peptides of the invention can be prepared by any technique known tothose skilled in the art or by techniques hereafter developed. Forexample, the peptides can be prepared using the solid-phase synthetictechnique (Merrifield, J. Am. Chem. Soc., 15:2149-2154 (1963); M.Bodanszky et al., (1976) Peptide Synthesis, John Wiley & Sons, 2d Ed.;Kent and Clark-Lewis in Synthetic Peptides in Biology and Medicine, p.295-358, eds. Alitalo, K., et al. Science Publishers, (Amsterdam, 1985).The synthesis of peptides by solution methods may also be used, asdescribed in The Proteins, Vol. II, 3d Ed., p. 105-237, Neurath, H., etal., Eds., Academic Press, New York, N.Y. (1976). The synthesizedpeptides may be substantially purified by preparative high performanceliquid chromatography or other comparable techniques available in theart. The composition of the synthetic peptides can be confirmed by antechnique for amino acid composition analysis.

In one embodiment, the peptides are provided as cyclic peptides.

For use in angiogenesis inhibition, prophylaxis and/or therapy of tumorsand/or metastasis, the peptides can be administered in a conventionaldosage form prepared by combining the peptides with a standardpharmaceutically acceptable carrier according to known techniques. Someexamples of pharmaceutically acceptable carriers can be found in:Remington: The Science and Practice of Pharmacy (2005) 21st Edition,Philadelphia, Pa. Lippincott Williams & Wilkins.

In one embodiment, the invention comprises at least one of the peptidesprovided by the invention in a pharmaceutical preparation, such as acomposition comprising at least one pharmaceutically acceptable carrier,which can be provided in a pharmaceutically acceptable bufferIn certainembodiments, compositions of the invention can include distinctpeptides, and can include mixtures of linear (non-cyclic) and cyclicpeptides.

Various methods known to those skilled in the art may be used tointroduce the compositions of the invention to an individual. Thesemethods include but are not limited to intracranial, intrathecal,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,oral, intranasal and retrograde routes.

It will be recognized by those of skill in the art that the form andcharacter of the particular dosing regime employed in the method of theinvention will be dictated by the route of administration and otherwell-known variables, such as rate of clearance, the size of theindividual and the stage of the particular disease being treated. Basedon such criteria, one skilled in the art can determine an amount of anyof the particular peptides described herein that will be effective toinhibit angiogenesis and/or tumor growth for any particular individual.It is generally considered that the amount of peptide administered willrange from microgram to milligram amounts.

The method of the invention can be performed in conjunction withconventional anti-cancer therapies. Such therapies can include but arenot limited to chemotherapies, such as androgen deprivation therapy,surgical interventions, and radiation therapy. The compositions of theinvention could be administered prior to, concurrently, or subsequent tosuch anti-cancer therapies.

The following Examples are meant to illustrate, but not limit theinvention.

Example 1

This Example demonstrates PSA-induced inhibition of gene expression inhuman prostate cancer cells.

The human prostate cancer cell lines LNCaP, DU-145 and PC-3 have low,moderate and high metastatic potential, and we reported that expressionof pro-angiogenic factors correlated with the metastatic potential(Aalinkeel R, et al., 2004. Cancer Res 64:5311-5321). Constitutiveexpression of multiple pro-angiogenic growth factors, including: VEGF,IL-8 and TGF-β, as well as MMP-9 and tissue inhibitors ofmetalloproteinase 1 and 4, was significantly greater in the highlymetastatic PC-3M cell line compared to LNCaP cells, with a low intrinsicmetastatic potential (Aalinkeel R, et al., 2004. Cancer Res64:5311-5321).

PSA purified to homogeneity from human seminal plasma is enzymaticallyactive, and free of contaminating hk2, and complexes with carrierproteins/chaperones (Bindukumar, B., et al., J Chromatogr B AnalytTechnol Biomed Life Sci, 2004. 813(1-2): p. 113-20). Gene expressionarray analysis of PC-3M cells versus LNCaP cells treated with 10 μM PSA,revealed that 136 genes were up-regulated and 137 genes weredown-regulated. QPCR analysis demonstrated that urokinase-typeplasminogen activator (uPA), VEGF, cysteine-rich angiogenic inducer-61,EphA2, TGF-β2, were significantly down-regulated in the highlymetastatic PC-3M cells, whereas, IFN-γ and several interferon relatedgenes, including 2,5 oligo-adenylated synthetase-2, were up-regulated(FIG. 1, top). The effect of PSA on VEGF and IFN-γ gene expression andprotein release in PC-3M cells was distinctly dose dependent(Bindukumar, B., et al., Neoplasia, 2005. 7(3): p. 241-52). Consistentwith the down-regulation of genes that stimulate tumor growth and induceangiogenesis in vitro summarized in FIG. 1, top, we demonstrated thatPSA inhibited growth of PC-3M xenografts in nude mice (FIG. 1, bottom,mean tumor volume 13 mm³ when PSA was administered in the tumorvicinity, compared to mean tumor volume in control animals, 267 mm³](Bindukumar, B., et al., Neoplasia, 2005. 7(3): p. 241-52).

Example 2

This Example provides an illustration of proteomic profiling of theeffect of PSA on human prostate cancer cells. In this regard, aproteomic approach was employed to identify proteins whose expressionwas modulated in PC-3M cells treated with PSA. 2D-DIGE coupled withHPLC-tandem mass spectrometry (MS/MS) identified a total of 41 proteinssignificantly changed (p<0.05) in abundance in PC-3M cells in responseto PSA treatment. Proteins from 26 gel spots were identified [FIG. 2].Many of down-regulated proteins, including: the N8 gene product longisoform, laminin receptor, vimentin, DJ-1 and Hsp60, are known to beinvolved in tumor progression. Specificity of PSA modulation ofdifferential expression of these genes in PC-3M cells was validated atthe gene level by RT-QPCR [FIG. 3].

Example 3

This Example demonstrates enzymatic activity of isolated PSA. Inparticular, enzymatic activity of PSA isolated from human seminal plasma(f-PSA) and from human prostate cancer tissue specimens (Tissue-PSA:T-PSA) was evaluated routinely using a substrate(Mu-His-Ser-Ser-Lys-Leu-Gln-AFC) (Calbiochem) that is highly specificfor PSA. In connection with the human prostate cancer tissue specimens,it should be noted that the majority of PSA in serum is complexed withserine protease inhibitors and is enzymatically inactive, whileessentially all of the PSA in seminal fluid is in a free form (f-PSA)and is enzymatically active. PSA in the tissue microenvironment (T-PSA)is largely free and enzymatically active. T-PSA levels are lower inadvanced prostate cancer than benign prostate, and T-PSA levelscorrelate with prognosis in prostate cancer, as well as in breastcancer; the higher the T-PSA level, the better the prognosis.

Hydrolysis of the fluorogenic substrate was quantitated using a LS-45Luminescence Spectrometer from Perkin Elmer) using the FL-Winlab^(R)program. Human prostate cancer tissue specimens were obtained from thePathology Resource Network at Roswell Park Cancer Institute under an IRBapproved protocol. Both f-PSA and T-PSA demonstrated enzymatic activityin the assay, however, T-PSA routinely demonstrated roughly 50% of theactivity of f-PSA from seminal plasma per microgram of protein, probablyreflecting loss of activity during extraction from tissue. Enzymaticactivity against the fluorescent substrate of both f-PSA and T-PSA wereinhibited completely by pre-incubation with zinc chloride (5 μM).

Example 4

This Example demonstrates that enzymatic activity of PSA is not requiredfor its effects on modulating angiogenesis related gene expression.

Enzymatically active PSA, and PSA inactivated by incubation with Zinc²⁺[activity inhibited >75% by 5.0 μM of Zinc, inset FIG. 4] were comparedfor their respective effects on expression of genes involved inangiogenesis/vasculogenesis in HUVEC cells. FIG. 5 demonstrates thatenzymatically active and inactive PSA were equally effective innegatively modulating expression of pro-angiogenic growth factors,including: bFGF and VEGF, as well as positively modulating expression ofIFN-γ, in HUVEC cells (FIG. 5), and expression of genes involved inblood vessel development such as FAK, FLT, KDR, TWIST1, Cathepsin D andP-38 in HUVEC cells (FIG. 6).

Example 5

This Example demonstrates the effect of enzymatically active f-PSA andT-PSA on endothelial tube formation in the Matrigel Assay.

FIG. 7 demonstrates the anti-angiogenic activity of PSA in the MatrigelEndothelial Tube Formation Assay. Equi-molar concentrations of bothf-PSA and T-PSA demonstrated equivalent inhibitory activity in this invitro assay of anti-angiogenic activity. Significantly, as demonstratedin the bar chart portion of FIG. 4, inhibition of endothelial tubeformation by f-PSA was not dependent on enzymatic activity. Over theentire range of PSA concentrations studied, PSA inactivated byincubation with Zn²⁺ was as inhibitory of tube formation as was nativePSA (data not shown in this figure).

Example 6

This Example demonstrates that illustrative PSA-mimetic peptidesprovided by the invention have inhibitory effect upon endothelial tubeformation by HUVEC in the Matrigel Assay. The 3-D model of human PSA,the amino acid sequence of the initial five candidate peptides, andtheir color-coded location on the exterior of the PSA protein moleculein aqueous solution, are presented in FIG. 8. Five PSA-mimetic peptideswith >90% purity were acquired from GeneScript Corporation, Piscataway,N.J., and tested for anti-angiogenic activity against HUVECs in theMatrigel tube formation assay with intact PSA as the control (FIGS. 9&10). Briefly, to perform these assays, 10⁴ HUVEC cells are mixed withthe PSA mimetic peptide, and, the mixture placed on top of a solidified,pre-poured Matrigel layer in a flat bottomed well of a multi-chamberedplate, and the culture incubated for 16-18 hr to allow time for theendothelial cells to migrate and form tubular structures. At theconclusion of the incubation, digital images were made from the centerof each culture to avoid bias, and 3-4 wells for each test conditionwere imaged. The images are processed under the Analyze 7.0 and“Angioquant” software packages, for imaging and quantification. Thetotal length of tubular structures in the image is determined, andaveraged over the replicate cultures. This method of quantificationallows for accurate, unbiased measurement of tube lengths. Peptideshaving each of the peptide sequences shown in FIG. 8 are encompassedwithin the scope of the invention, including all modifications andsubstitutions to the sequences as described supra. Likewise, all methodsof the invention described herein pertain to the peptide sequences shownin FIG. 8.

FIG. 9 shows the dose-related effect on angiogenesis (endothelial tubeformation in Matrigel) (average of 5 individual determinations perexperiment) for the five synthetic, PSA-mimetic peptides, at twodifferent concentrations (10 and 100 μM), and enzymatically activenative PSA (10 μM) as a positive control. The experimental endpoint,measured by digital image analysis, is the total area of the imagecomprised of endothelial cells (tubes). Analysis of total length oftubular structures, as well as area covered by tubular structures,produced comparable findings. The maximal inhibition of tubule formationwas seen with Peptide #1 (amino acids 107-119 of PSA) at a concentrationof 100 μM, and peptide #3 (PSA amino acids 181-194) at a concentrationof 10 μM. While the reason for the decrease in the amount ofanti-angiogenic activity in this figure when the concentration of thepeptides is increased from 10 to 100 μM is not clear, it may be relatedto a dosing response observed in this case. As noted above, Peptides #2,#4 and #5 did not demonstrate reproducible anti-angiogenic activity,demonstrating that the peptides of the invention were not obvious designchoices. FIG. 10 presents the evaluation of anti-angiogenic activity forpeptides #1 and #3 relative to the level of inhibition by native PSA,averaged over multiple experiments with different parental populationsof HUVEC cells. Across multiple sets of experiments, both peptidesinhibited the angiogenic activity of HUVEC by 35-40% relative to nativePSA.

Example 7

The stability of a peptide against proteolytic degradation is animportant factor for the use of peptides in vivo. Peptides arefrequently modified to prevent enzymatic degradation. Several approacheshave been used to achieve peptide stability including use of D-aminoacids, peptidomimetics or cyclization. For this Example we prepared acyclic variant of peptide #3 and compared the biological activities ofthe linear and cyclic peptide. Peptide #3 (¹⁸¹Asn Asp Val Cys Ala GlnVal His Pro Gln Lys Val Thr Lys) (SEQ ID NO:3) had one cysteine residue(#184) in its structure and it was replaced with alanine duringsynthesis. It is demonstrated that linear peptide #3 at 10 μMconcentration significantly inhibits HUVEC tube formation in Matrigel(FIGS. 9&10). The single Cys in Peptide #3 was replaced to avoidpotential disulfide formation with distinct proteins or peptides.Peptide #6 has essentially the same sequence as Peptide #3 with twoexceptions. The sequence was extended to include five additional aminoacid residues towards the carboxyl end in order to include anothernaturally existing cysteine (residue #198) in the sequence. Bothcysteins (#184 & 198) in this newly synthesized cyclic peptide were notmodified and remained in their natural position (¹⁸¹Asn Asp Val Cys AlaGln Val His Pro Gln Lys Val Thr Lys Phe Met Leu Cys Ala). (SEQ ID NO:6).As in case of linear peptide, this cyclic peptide was also acetylated atN-terminus and amidated at C-terminus.

Linear and cyclic peptides were evaluated for their ability of inhibitHUVEC tube formation in an antiangiogenic assay in Matrigel. The resultsare shown in FIG. 11 and demonstrate that cyclization of peptide #3 didnot alter its physiological properties and inhibited HUVEC tubeformation even more efficiently than linear peptide at certainconcentrations.

Example 8

The following Example provides a description of our characterization oftwo putative binding sites for PSA on receptor(s) of Human UmbilicalVein Endothelial cells. Illustrative mass spectroscopy results are shownin FIG. 12. To obtain the data described in this Example and thosesummarized in FIG. 12 the following materials and methods were used.

Biotinylation of f-PSA

The procedure for biotin labeling of proteins is known in the art. Inbrief, 10 mM of Sulfo-NHS-LC-Biotin (Pierce, Rockford, Ill.) wasdissolved in PBS, pH 7.4 and added in 20-fold molar excess to 5 mg off-PSA and incubated at room temperature for 30 min. Excess reactivebiotin was quenched by the adding 1M Tris, pH 7.2 at 1/10th the volumeof the biotin labeling reaction, and incubated at room temperature for10 mM Biotinylated PSA was separated from excess biotin bycentrifugation at 2000 rpm using a concentrator with a 10,000 MW cut offmembrane (Millipore Corp., Billerica, Mass.). The labeled PSA wasaliquoted in 0.5 mL volumes and frozen at −80° C. Biotinylated PSA wasdetected by ELISA using anti-PSA antibodies and by Western Blot usingHRP-conjugated Streptavidin (Pierce, Rockford, Ill., USA).

Biotinylated PSA and Isolation of PSA-Target Complexes

The details of the protocol for isolation of receptor targets usingbiotinylated PSA and crosslinking approach are as follows: Approximately30×10⁶ HUVECs were grown on petri dishes (10 cm, BD Falcon, FranklinLakes, N.J.) to 90% confluency. Media was removed and the cells werewashed once with PBS. Cells were incubated with serum free mediacontaining 10 μM biotin labeled PSA and 1:100 (v/v) protease inhibitorcocktail (Sigma, St. Louis, Mo.) for 15 minutes at 4° C. The bindingsolution was removed and 25 mM of dithiobis[succinimidyl-propionate](DSP) crosslinker, dissolved in DMSO, was added at final concentrationof 10 mM to each dish and incubated for 30 min at room temperature. Thecrosslinking reaction was stopped by adding 1M Tris, pH 7.2 for a finalconcentration of 20 mM for 15 minutes at room temperature. Cells wereremoved from the dishes by using a cell scraper (Costar, Corning, N.Y.)and washed with PBS. Cells collected were centrifuged at 1000 rpm andthe supernatant discarded. The cell pellet was lysed using mammalianprotein extraction reagent (M-PER) (Pierce, Rockford, Ill.) containing1:100 (v/v) protease inhibitor cocktail for 15 minutes at roomtemperature and then centrifuged at 14,000 rpm for 10 minutes. Thesupernatant was collected to a new tube for further processing. Toisolate the PSA-target complex, the cell debris pellet was mixed withnon-reducing loading buffer or NP-40 (Sigma, St. Louis, Mo.) at a 1:1(v/v) ratio and incubated in a 60° C. water bath to re-solubilizemembrane proteins, which may contain the biotin labeled PSA crosslinkedto the target molecule(s). The supernatant was collected and run onSDS/PAGE, western blot analysis, and probed with streptavidin-HRP forthe PSA-target complex. Based on the presence of a band higher than 33kDa, the re-solubilized sample was run on SDS/PAGE, stained with DeepPurple Total Protein Stain (GE Healthcare Life Sciences, Piscataway,N.J.), the band of interest was cut out, and sent for mass spectroscopyanalysis.

Trypsin Digestion

The solubilized cellular fraction was run under non-reducing conditionson an SDS-PAGE gel, stained with Deep Purple Total Protein Stain (GEHealthcare, Piscataway, N.J.) and scanned using a Typhoon 9410 Imager(GE Healthcare, Piscataway, N.J.). Protein bands of interest wereexcised manually and placed in 0.6 mL microtube (Axygen, pre-washed with18MΩ·cm water (Milli-Q) and methanol). In-gel trypsin digestion wasperformed according to standard operating procedures routinely used inthe Roswell Park Cancer Institute

Proteomics Facility. In brief, gel pieces were de-stained with 200 μL50% acetonitrile/100 mM ammonium bicarbonate solution for 30 minuteswith constant mixing (MixMate, Eppendorf, Hauppauge, N.Y.). Afterremoval of the de-stain solution, gel pieces were dehydrated in 100 μLacetonitrile for 15 minutes at room temperature (RT) and dried in aSpeedvac concentrator (Eppendorf, Hauppauge, N.Y.). Dried gel pieceswere reduced with 10 mM DTT (Sigma, St. Louis, Mo.)/100 mM sodiumbicarbonate solution at RT for 45 minutes. After removal of thissolution, samples were alkylated with 200 μL 50 mM iodoacetamide (Sigma,St. Louis, Mo.)/100 mM sodium bicarbonate at RT for 30 minutes underdark conditions. After removal of alkylation solution, samples werewashed with 200 μL of 100 mM sodium bicarbonate and incubated with 200μL of 50% acetonitrile/100 mM sodium bicarbonate at RT for 10 minutes.Samples were dehydrated with 100 μL acetonitrile and dried in a Speedvacconcentrator and digested with trypsin (Promega, Madison, Wis., 10 ng/μLin 10% acetonitrile/40 mM sodium bicarbonate, 30-50 μL) at 37° C. for 16hours. The digests were extracted twice with 100 μL 50%acetonitrile/0.1% TFA at RT for 60 minutes with constant mixing. Theextracts were pooled and dried in a Speedvac concentrator and eachsample was then reconstituted with 8 μL of 2% formic acid (FA).

LC-MS/MS Analysis

Trypsin digested samples (5 μL each) were analyzed by LCnanoelectrospray-tandem mass spectrometry (LC-ESI-MS/MS) using ananoACQUITY UPLC (Waters Corp, Milford, Mass.) coupled through anebulization-assisted nanospray ionization source to a Q-ToF Premiermass spectrometer (Waters/Micromass, Milford, Mass.) The LC consisted ofa trap column (Symmetry C18, 5μ, 180μ×20 mm, Waters, Milford, Mass.),followed by separation on an analytical column (Atlantis C18, 3μ,100μ×10 cm, Walters, Milford, Mass.). Samples were loaded, trapped, andwashed at a flow rate of 3 μL/min with 98% solvent A (water containing0.1% FA)/2% solvent B (acetonitrile containing 0.1% FA) for 5 minutes.Peptides were eluted with a gradient of 98% A/2% B to 40% A/60% B for 40minutes at 0.4 μL/min, 10% A/90% B for 7 minutes at 1.5 μL/minute, andthen 10% A/90% B at 1.5 μL/minute for 8 minutes. Throughout thegradient, the mass spectrometer was programmed (Data DependentAcquisition experiment, DDA) to monitor ions with m/z in the range of300-1500, and ions with +2 to +4 charges only were selected for MS/MSexperiments using the preset DDA collision energy parameters.

Database Search and Peptide and Protein Identification

MS/MS spectra were processed and transformed to the PKL file formationusing Proteinlynx Global Server v2.3 (Waters/Micromass, Milford, Mass.)and the default parameters of MaxEnt3 (Waters/Micromass, Milford,Mass.). The PKL files were used to search the Homo sapiens subset of theSwiss-Prot database (containing 20,352 sequences) using a locallyinstalled version of MASCOT (Matrix Science, v 2.2.2). The searchparameters were as follows: trypsin as the proteolytic enzyme with 2possible missed cleavages, carboxyamidomethylation of cysteine as afixed modification, NHS-LC-Biotin of lysine, and3-(carbamidomethylthio)propanoyl of lysine as a variable modification,the allowable mass error was 100 ppm for peptides and 100 mDa forfragment ions, peptide charge was set to 2+ and 3+, the instrument wasset to ESI-QUAD-TOF. The mascot default significance threshold of p<0.05for assignments was used in the searches and a minimum of two uniquepeptides were used as a criteria for a match. This example suggeststhere is/are proteins on the cell membrane that are specific bindingsites for intact PSA and that these receptors modulate the biologicaleffect of PSA.

Example 9

The following materials and methods were used in obtaining some of thedata presented herein.

Fmoc-Based Solid-Phase Peptide Synthesis: Peptides were prepared bymanual solid-phase peptide synthesis. Fmoc groups for Na protection werecleaved by 8 min treatment with 20% piperidine in DMF followed by thesecond treatment with the same reagent for 10 min. After the Fmoccleavage, the rink-resin was washed with DMF (×6). The next residue wasthen incorporated with the DIPC/HOBt coupling protocol [Fmoc-amino acid(3 equiv), DIPC (3 equiv), and HOBt (3 equiv)]. After gentle agitation(1 hr) and washing with DMF (×6), part of the peptide-resin wassubjected to the Kaiser test. On completion of the assembly, thepeptide-resin was successively washed with DMF (×3), DCM (×4) and thendried in vacuo.

Cleavage and Deprotection. The cleavage cocktail (Trifluoroaceticacid/H2O=95/5, v/v; 3 ml/100 mg of resin) is added to the washed resin.Complete deprotection was achieved in the cocktail in 4 hrs at 30° C.Following the cleavage reaction, TFA was removed by evaporation.

Disulfide Bond Formation. After cleaved from resin, the crude linearpeptide was obtained, which was characterized by RP-HPLC and Maldi-TOFMS. Disulfide formation was realized by oxidation in air (pH˜8), whichwas monitored by RP-HPLC, MS and free sulfhydryl detection (DTNBmethod).

Purification by Preparative RP-HPLC and Purity Assessment by AnalyticalHPLC. A crude peptide sample was purified by preparative RP-HPLC(HP1100, Agilent) using a column of Daiso C18 (10 μm, 100 Å, 50×250 mm).A solvent system consisting of solvent A (0.05% TFA, 2% CH3CN in water)and solvent B (90% CH3CN/H2O) at a flow rate of 25 mL/min was used forelution, and the absorbance was detected at 220 nm. The solvent wasremoved by lyophilization to afford a fluffy powder as a final purifiedpeptide. The chemical structure was characterized by MALDI-TOF-MS, andthe purity of the purified material was assessed by analytical HPLC(C18-4.6×250 mm, flow rate of 1 mL/min), and the absorbance was detectedat 220 nm

Example 10

Using peptides made and tested as described above, the data presented inFIGS. 13-21 were obtained. For all these figures, the peptideconcentration is 10 uM.

FIG. 13 is a graphical representation of data showing anti-angiogenicactivity of f-PSA, PSA-P3L, and PSA-P3C by inhibiting tube formation byHUVEC in a Matrigel assay. To produce the results summarized in FIG. 13,a 24-well tissue culture plate was coated with 200 ul/well of Matrigeland incubated for 30 min at 37 C. Approximately 80,000 HUVEC in 0.5 mlof media supplemented with f-PSA or PSA-P3L, PSA-P3C was applied on topof the Matrigel layer and cells were incubated for 18 hr at 37 C foeendothelial cell tube formation. Live cell images (4× magnification)were taken using Nikon Eclipse TE300 inverted microscope system andanalyzed using Spot Advance software program. Five images weretaken/well. The images were processed further using Analyze 7.0(AnalyzeDirect, Inc., Overland Park, Kans.) and Angioquant v1.33 toobtain the average tube length for each image. Percent inhibition isexpressed in relation to tube length in untreated control cells.

FIG. 14 is a graphical representation of data showing of f-PSA, PSA-ACTComplex, PSA-P3L, and PSA-P3L-ACT complex by inhibiting tube formationby HUVEC in a Matrigel assay. As can be seen from this Figure, f-PSA hassignificant anti-angiogenic activity. However, there is a significantloss of anti-angiogenic activity of f-PSA after it complexes with alpha1anti-chymotrypsin (ACT). On the other hand there is no loss ofanti-angiogenic of PSA-P3L in the presence of ACT. The details ofanti-angiogenic assay are identical as described above.

FIG. 15 is a graphical representation of data showing modulation of FLT1gene expression in HUVEC by f-PSA and PSA-P31. As can be seen from thisFigure, f-PSA and PSA-P3L effectively inhibit FLT1 gene expression inHUVEC. To obtain the data summarized in this figure, nearly confluentmonolayers of HUVEC were treated with 10 uM of f-PSA or PSA-P3L; RNAextracted and reverse transcribed. The c-DNA was amplified by Real-timeQPCR using specific primer. The results shown are average of threeexperiments.

FIG. 16 is a graphical representation of data showing modulation of FAKgene expression in HUVEC by f-PSA and PSA-P3L. To produce the resultssummarized in FIG. 16, nearly confluent monolayers of HUVEC were treatedwith 10 uM of f-PSA or PSA-P3L; RNA extracted and reverse transcribed.The c-DNA was amplified by Real-time QPCR using specific primers. Theresults shown are the average of three experiments. As can be seen fromthis figure, 16 f-PSA and PSA-P3C significantly down-regulates FAK geneexpression in HUVEC. PSA-P3L and control were not significantlydifferent which indicates that cyclic peptide is more effective thanlinear peptide. The other experimental details are similar to all geneexpression studies. Thus, in one embodiment, the invention provides amethod for inhibiting FAK gene expression in human endothelial cellscomprising contacting the cells with one or more peptides of theinvention.

FIG. 17 is a graphical representation of data showing modulation of VEGFgene expression in HUVEC by f-PSA, PSA-P3L and PSA-P3C. To produce theresults summarized in FIG. 17, nearly confluent monolayers of HUVEC weretreated with 10 uM of f-PSA or PSA-P3L; RNA extracted and reversetranscribed. The c-DNA was amplified by Real-time QPCR using specificprimers. The results shown are average of three experiments. PSA-P3L wasnot significantly different from control. The data represents an averageof three experiments. As can be seen from this figure, PSA-P3C and f-PSAsignificantly down-regulate VEGF gene expression in HUVEC. Thus, in oneembodiment, the invention provides a method for inhibiting VEGFexpression in human endothelial cells comprising contacting the cellswith one or more peptides of the invention.

FIG. 18 is a graphical representation of data showing modulation of ANG2gene expression in HUVEC by f-PSA, PSA-P31 and PSA-P3C. To produce theresults summarized in FIG. 18, nearly confluent monolayers of HUVEC weretreated with 10 uM of f-PSA, PSA-P3L or PSA-P3C; RNA extracted andreverse transcribed. The c-DNA was amplified by Real-time QPCR usingspecific primer. The results shown are an average of three experiments.As can be seen from this figure, f-PSA and PSA-P3C significantlyinhibited ANG2 gene expression whereas PSA-P3L was not significantlydifferent from control. Thus, PSA-P3C and f-PSA significantlydown-regulate ANG2 gene expression in HUVEC. Accordingly, in oneembodiment, the invention provides a method for inhibiting ANG2expression in human endothelial cells comprising contacting the cellswith one or more peptides of the invention.

FIG. 19 is a graphical representation of data showing anti-angiogenicactivity of f-PSA, PSA-P1L and PSA-P3L via inhibition of tube formationby HUVEC in Matrigel. As can be seen from this figure, linear peptidesPSA, PSA-P1L and PSA-P3L significantly inhibit HUVEC tube formation inan in vitro anti-angiogenic assay.

FIG. 20 is a graphical representation of data showing anti-angiogenicactivity of f-PSA and PSA-ACT complexes via inhibition of tube formationby HUVEC in Matrigel. As can be seen from this figure, f-PSA hassignificant anti-angiogenic activity. In in vitro anti-angiogenic assay,f-PSA inhibits HUVEC tube formation in Matrigel. However thisphysiological activity of PSA is significantly reduced when it complexeswith serine protease inhibitor, alpha 1, anti-chymotrypsin (ACT). Theresults shown here are an average of three experiments.

FIG. 21 is photographic representation of the effect of f-PSA, PSA-P1L,PSA-P1C, PSA-P3L and PSA-P3C on migratory properties of HUVEC. Toproduce the results summarized in FIG. 21, a commercially availableCytoSelect 24-well Wound Healing Assay kit was used. Monolayers weredisrupted by the plastic insert to produce a linear wound (0.9 mmwidth), the wells washed with PBS to remove debris, and culturesincubated with f-PSA, PSA-P11, PSA-P1C, PSA-P3L or PSA-P3C at 10 uMconcentration. Wounded fields were measured at 48 hr using phasecontrast microscopy to measure wound size.

The foregoing description of specific embodiments is for the purpose ofillustration and is not to be construed as restrictive. From theteachings of the present invention, those skilled in the art willrecognize that various modifications and changes may be made withoutdeparting from the spirit of the invention.

We claim:
 1. A peptide having a sequence selected from Lys Asn Arg PheLeu Arg Pro Gly Asp Asp Ser Ser His (SEQ ID NO:1), Cys Lys Asn Arg PheLeu Arg Pro Gly Asp Asp Ser Ser His Cys (SEQ ID NO:7), Asn Asp Val CysAla Gln Val His Pro Gln Lys Val Thr Lys (SEQ ID NO:3), Val Ala Ala GlnVal His Pro Gln Lys Val Thr Lys (SEQ ID NO:8), Asn Asp Val Cys Ala GlnVal His Pro Gln Lys Val Thr Lys Phe Met Leu Cys Ala (SEQ ID NO:6), andAsn Asp Val Ala Ala Gln Val His Pro Gln Lys Val Thr Lys Phe Met Leu AlaAla (SEQ ID NO:9).
 2. The peptide of claim 1, wherein the peptide iscyclic.
 3. The peptide of claim 2, wherein the peptide has the sequenceof SEQ ID NO:6, and wherein the two Cys in the sequence of SEQ ID NO:6are connected to one another via a disulfide bond, or wherein thepeptide has the sequence of SEQ ID NO:7, and wherein the two Cys in thesequence of SEQ ID NO:7 are connected to one another via a disulfidebond.
 4. The peptide of claim 1, wherein the peptide comprises one, two,three, or four conservative amino acid substitutions, and wherein thepeptide is capable of inhibiting tubule formation by human umbilicalvein endothelial cells.
 5. The peptide of claim 1, wherein the peptideis present in a composition comprising a pharmaceutically acceptablecarrier.
 6. The peptide of claim 5, wherein the peptide is cyclic. 7.The composition of claim 6, wherein the isolated peptide has thesequence of SEQ ID NO:6, and wherein the two Cys in the sequence of SEQID NO:6 are connected to one another via a disulfide bond, or whereinthe isolated peptide has the sequence of SEQ ID NO:7, and wherein thetwo Cys in the sequence of SEQ ID NO:7 are connected to one another viaa disulfide bond.
 8. A method of inhibiting angiogenesis in anindividual comprising administering a composition comprising a peptideof claim 1 to an individual in an amount effective to inhibit theangiogenesis in the individual.
 9. The method of claim 8, wherein theinhibition of angiogenesis comprises inhibition of angiogenesis in atumor in the individual.
 10. The method of claim 8, wherein the peptideis cyclic.
 11. The method of claim 10, wherein the peptide has thesequence of SEQ ID NO:6, and wherein the two Cys in the sequence of SEQID NO:6 are connected to one another via a disulfide bond, or whereinthe isolated peptide has the sequence of SEQ ID NO:7, and wherein thetwo Cys in the sequence of SEQ ID NO:7 are connected to one another viaa disulfide bond.
 12. The method of claim 8, wherein the compositioncomprises more than one peptide of claim
 1. 13. A method of inhibitinghuman endothelial cell migration comprising contacting human endothelialcells with a composition comprising a peptide of claim 1, wherein themigration is inhibited subsequent to the contacting of the humanendothelial cells with the composition.
 14. The method of claim 13,wherein the peptide is cyclic.
 15. The method of claim 14, wherein thepeptide has the sequence of SEQ ID NO:6, and wherein the two Cys in thesequence of SEQ ID NO:6 are connected to one another via a disulfidebond, or wherein the isolated peptide has the sequence of SEQ ID NO:7,and wherein the two Cys in the sequence of SEQ ID NO:7 are connected toone another via a disulfide bond.